Angiogenesis, the formation of new capillary blood vessels, is essential for the growth and metastasis of solid tumors. The Vascular Endothelial Growth Factor (VEGF) family of proteins is one of the most potent and specific positive regulators of angiogenesis. These proteins bind to three tyrosine-kinase receptors, VEGFR-1 (Vascular Endothelial Growth Factor Receptor 1/Flt-1), VEGFR-2 (KDR/Fetal liver kinase 1, Flk-1) and VEGFR-3 (Flt-4). Neuropilin-1 is the fourth receptor that can bind specifically one of the VEGF isoforms: (VEGF165) and enhances its binding to VEGFR-1 (Soker et al., Cell 92(6):735-45, 1998). In general, VEGFR-1 and VEGFR-2/KDR are expressed on vascular endothelial cells whereas VEGFR-3 is expressed on lymphatic endothelial cells (Partanen et al., Cancer 86:2406, 1999; Kaipainen et al., Proc Natl Acad Sci USA 92:3566-70, 1995).
VEGFR-2/KDR is a major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF (Ferrara et al., Nat. Med. 9(6):669-676, 2003), and VEGFR-2/KDR is involved in the process of vascularization and angiogenesis. For example VEGFR-2/KDR-null mice die in utero between days 8.5 and 9.5 without any sign of vasculogenesis or organized blood vessels (Shalaby et al. Nature 376:62-66, 1995), demonstrating that VEGFR-2/KDR has an important role in the process of vascularization and angiogenesis.
VEGFR-2/KDR is highly expressed in tumor associated endothelial cells and contributes to tumor growth, invasion and metastasis (Dias et al., J Clin Invest. 106(4):511-521, 2000; Santos et al., Blood 103(10):3883-3889, 2004; St. Croix et al., Science 289:1197-1202, 2000). In addition, VEGFR-2/KDR is also expressed on the surface of several tumor cells including: B cell lymphoma and leukemia, multiple myeloma, urothelial bladder cancer, breast cancer, and lung cancer, among others (El-Obeid et al., Leuk Res. 28(2): 133-137, 2004; Kumar et al., Leukemia 17(10):2025-2031, 2003; Gakiopoulou-Givalou et al., Histopathology 43(3):272-279, 2003; Kranz et al., Int J Cancer 84(3):293-298, 1999; Decaussin et al., J Pathol. 188(4):369-377, 1999). The relatively high level of expression on tumor cells relative to normal vascular endothelial cells suggests that VEGFR-2/KDR is a suitable target of tumor therapy.
Anti-angiogenic strategies targeting VEGFR-2/KDR have long been sought. Small molecule tyrosine kinase inhibitors targeting VEGFR-2/KDR, such as SU5416 have been shown to be effective in treating certain types of carcinomas in vitro and in vivo (Fong et al., Cancer Res. 59(1):99-106, 1999; Zangari et al., Clin Cancer Res. 10(1 Pt 1):88-95, 2004). Monoclonal antibodies against VEGFR-2/KDR can inhibit tumor angiogenesis and growth of several human and murine tumors (Prewett et al., Cancer Res. 59(20):5209-5218, 1999; Zhu et al., Cancer Res. 58(15):3209-3214, 1998). Wei et al., have shown that specific immune responses can be induced by xenogeneic endothelial cells and can protect mice from tumor challenge (Wei et al., Nat. Med. (10):1160-1166, 2000). Vaccine strategies against VEGFR-2/KDR have also been attempted. Tumor growth can be successfully inhibited by immunotherapy targeting VEGFR-2/KDR through a DNA vaccine encoding the full-length VEGFR-2/KDR (Niethammer et al., Nat Med 8:1369-1375, 2002), or dendritic cells transfected with a full-length VEGFR-2/KDR encoding construct (Nair et al., Blood 102(3):964-971, 2003), or pulsed with recombinant full-length VEGFR-2/KDR protein (Li et al., J. Exp. Med. 195(12):1575-1584, 2002). Some of these strategies have attributed the anti-tumor effects elicited to CD8+CTLs (Wei et al., Nat. Med. (10): 1160-1166, 2000; Niethammer et al., Nat Med 8:1369-1375, 2002; Yiwen et al., J. Exp. Med. 195:1575-1584, 2002), indicating that CTLs elicited by immunization against VEGFR-2/KDR can destroy endothelial cells derived from tumor associated vessels. To date, no human or murine VEGFR-2/KDR CTL epitopes have been identified.
The present disclosure provides murine and human MHC Class I epitopes of VEGFR-2/KDR, and demonstrates their efficacy as an anti-angiogenic vaccine.